Surface plasmon resonance (SPR) based biosensors are commonly used to perform kinetic studies of complex molecular interactions such as between hormone-receptor, enzyme-substrate and antigen-antibody. The biosensors are typically in the form of one or more sensing regions housed within a flow cell of a microfluidic system. The microfluidic system defines a series of flow paths that direct fluid flow to the flow cell containing the sensing regions. The one or more sensing regions of the flow cell support immobilized molecules referred to as “ligands.” The ligands bind molecules known as “analytes” which are present in fluids that are directed to the sensing region via the microfluidic system. Current analysis methods determine the kinetic interaction of the ligand and analyte by separately injecting a series of analyte concentrations into the system and measuring the change in refractive index at the sensing regions. Based on the changes in refractive index, one can determine the real-time kinetics of the interaction between the analyte and ligand, including association and dissociation rates.
When the analyte being tested consists of a small molecule, the change in refractive index at the sensing region due to interaction with ligand is typically very small and may be difficult to detect above the refractive index from the bulk flow of the sample in the sensing region (bulk refractive index). This is typically a result of high concentrations of the small molecule or more commonly, high concentrations of solubilizing agent such as dimethylsulfoxide (DMSO). As such, a referencing method is needed that allows the response from the bulk refractive index to be separated from the response due to the molecular interaction between analyte and immobilized ligand at the sensing region.
The double referencing method has been one approach to solving this problem. In this method, the sample is caused to flow over two separate sensing regions; one of the sensing region contains immobilized ligand (working sensing region) and one sensing region is free of immobilized ligand (reference sensing region). Thus, the response from the reference sensing region can be subtracted from the response in the working sensing region to yield the response attributed to the molecular interaction in the working sensing region.
However, there are some limitations associated with this particular method. Differences, such as temperature, sensitivity and dispersion, between the working sensing region and reference sensing region can significantly impact the data quality and obscure the binding response, especially with low molecular weight molecules. Dispersion of the sample that may occur between the two sensing regions is of a particular concern. In most microfluidic systems, a buffer fluid will typically flow through the channels housing the sensing regions prior to exposure to the fluid containing the analyte sample. Since the sensing regions are separated, the sample is likely to encounter additional residual buffer in the flow path from the reference sensing region to the working sensing region. This causes dilution of the sample thereby changing the refractive index such that the response obtained from the reference sensing region is not entirely an accurate representation of the sample that encounters the working sensing region. These concerns are particularly pertinent when the bulk refractive index of the sample and buffer differ, even where the difference is small (e.g. 500 RU). These small differences can have profound effects on the accuracy of the kinetic calculations for the association and dissociation rates of the binding interactions between analyte and ligand. Thus, a method is needed that provides a reference measurement without the deleterious effects of a two sensing region referencing system.